Active Site Tyrosine Research Articles

Structural insights into the assembly of type IIA topoisomerase DNA cleavage-religation center.

The ability to catalyze reversible DNA cleavage and religation is central to topoisomerases' role in regulating DNA topology. In type IIA topoisomerases (Top2), the formation of its DNA cleavage-religation center is driven by DNA-binding-induced structural rearrangements. These changes optimally position key catalytic modules, such as the active site tyrosine of the WHD domain and metal ion(s) chelated by the TOPRIM domain, around the scissile phosphodiester bond to perform reversible transesterification. To understand this assembly process in detail, we report the catalytic core structures of human Top2α and Top2β in an on-pathway conformational state. This state features an in trans formation of an interface between the Tower and opposing TOPRIM domain, revealing a groove for accommodating incoming G-segment DNA. Structural superimposition further unveils how subsequent DNA-binding-induced disengagement of the TOPRIM and Tower domains allows a firm grasp of the bound DNA for cleavage/religation. Notably, we identified a previously undocumented protein-DNA interaction, formed between an arginine-capped C-terminus of an α-helix in the TOPRIM domain and the DNA backbone, significantly contributing to Top2 function. This work uncovers a previously unrecognized role of the Tower domain, highlighting its involvement in anchoring and releasing the TOPRIM domain, thus priming Top2 for DNA binding and cleavage.

Replacement of Tyrosines by Unnatural Amino Acid Aminophenylalanine Leads to Metal-Mediated Aniline Free Radical Formation in a Copper Amine Oxidase.

Copper amine oxidases (CAOs) catalyze the oxidative deamination of primary amines to aldehyde, ammonia, and hydrogen peroxide as products and are widely distributed in bacteria, plants, and eukaryotes. These enzymes initiate the single turnover, post-translational conversion of an active site tyrosine to the redox cofactor 2,4,5-trihydroxyphenylalanine quinone (TPQ), subsequently employing TPQ to catalyze steady-state amine oxidation. The mechanisms of TPQ biogenesis and steady-state amine oxidation have been studied extensively, with consensus mechanisms proposed for both reactions. One unresolved issue has been whether the Cu2+ center must undergo formal reduction to Cu1+ in the course of the reaction. Herein, we investigate the properties of the active site of a yeast (Hansenula polymorpha) amine oxidase (HPAO) that has undergone site-specific insertion of a para-aminophenylalanine (pAF) into the position of either the precursor tyrosine to TPQ (Y405) or the two strictly conserved neighboring tyrosines (Y305 and Y407). While our original intention was to interrogate cofactor biogenesis using a precursor unnatural amino acid (UAA) of altered redox potential and pKa, we instead observe an unanticipated reaction assigned to an intramolecular electron transfer from pAF to the active site copper ion. We establish the generality of the observed active site chemistry using exogenously added, aniline-containing substrates under conditions that prevent side chain amine oxidation. The results support previous proposals that the activation of the TPQ precursor occurs in the absence of a formal valence change at the active site copper site. The described reaction of pAFs with the active site redox Cu2+ center of HPAO provides a prototype for either the engineering of the enzymatic oxidation of exogenous anilines or the insertion of site-specific free radical probes within proteins.

Rapid, DNA-induced interface swapping by DNA gyrase

DNA gyrase, a ubiquitous bacterial enzyme, is a type IIA topoisomerase formed by heterotetramerisation of 2 GyrA subunits and 2 GyrB subunits, to form the active complex. DNA gyrase can loop DNA around the C-terminal domains (CTDs) of GyrA and pass one DNA duplex through a transient double-strand break (DSB) established in another duplex. This results in the conversion from a positive (+1) to a negative (–1) supercoil, thereby introducing negative supercoiling into the bacterial genome by steps of 2, an activity essential for DNA replication and transcription. The strong protein interface in the GyrA dimer must be broken to allow passage of the transported DNA segment and it is generally assumed that the interface is usually stable and only opens when DNA is transported, to prevent the introduction of deleterious DSBs in the genome. In this paper, we show that DNA gyrase can exchange its DNA-cleaving interfaces between two active heterotetramers. This so-called interface ‘swapping’ (IS) can occur within a few minutes in solution. We also show that bending of DNA by gyrase is essential for cleavage but not for DNA binding per se and favors IS. Interface swapping is also favored by DNA wrapping and an excess of GyrB. We suggest that proximity, promoted by GyrB oligomerization and binding and wrapping along a length of DNA, between two heterotetramers favors rapid interface swapping. This swapping does not require ATP, occurs in the presence of fluoroquinolones, and raises the possibility of non-homologous recombination solely through gyrase activity. The ability of gyrase to undergo interface swapping explains how gyrase heterodimers, containing a single active-site tyrosine, can carry out double-strand passage reactions and therefore suggests an alternative explanation to the recently proposed ‘swivelling’ mechanism for DNA gyrase (Gubaev et al., 2016).

Rapid, DNA-induced interface swapping by DNA gyrase.

DNA gyrase, a ubiquitous bacterial enzyme, is a type IIA topoisomerase formed by heterotetramerisation of 2 GyrA subunits and 2 GyrB subunits, to form the active complex. DNA gyrase can loop DNA around the C-terminal domains (CTDs) of GyrA and pass one DNA duplex through a transient double-strand break (DSB) established in another duplex. This results in the conversion from a positive (+1) to a negative (-1) supercoil, thereby introducing negative supercoiling into the bacterial genome by steps of 2, an activity essential for DNA replication and transcription. The strong protein interface in the GyrA dimer must be broken to allow passage of the transported DNA segment and it is generally assumed that the interface is usually stable and only opens when DNA is transported, to prevent the introduction of deleterious DSBs in the genome. In this paper, we show that DNA gyrase can exchange its DNA-cleaving interfaces between two active heterotetramers. This so-called interface 'swapping' (IS) can occur within a few minutes in solution. We also show that bending of DNA by gyrase is essential for cleavage but not for DNA binding per se and favors IS. Interface swapping is also favored by DNA wrapping and an excess of GyrB. We suggest that proximity, promoted by GyrB oligomerization and binding and wrapping along a length of DNA, between two heterotetramers favors rapid interface swapping. This swapping does not require ATP, occurs in the presence of fluoroquinolones, and raises the possibility of non-homologous recombination solely through gyrase activity. The ability of gyrase to undergo interface swapping explains how gyrase heterodimers, containing a single active-site tyrosine, can carry out double-strand passage reactions and therefore suggests an alternative explanation to the recently proposed 'swivelling' mechanism for DNA gyrase (Gubaev et al., 2016).

Tern-701 (HS-10382) Is a Potent Inhibitor of BCR::ABL1 and Is Synergistic with Active Site Tyrosine Kinase Inhibitors

CONCLUSIONS Although tyrosine kinase inhibitors (TKIs) targeting the ATP-binding site of the BCR::ABL1 oncoprotein are effective therapeutics for chronic myeloid leukemia (CML), patients often develop drug resistance due to ATP-site mutations that inhibit drug binding. TERN-701 is a potent and highly selective inhibitor of BCR::ABL1 that is designed to bind the allosteric myristoyl site on the kinase, circumventing resistance to active site mutations, with potential for synergistic combination with active site TKIs. Competition binding screens were used to assess the selectivity of TERN-701 on more than 450 targets spanning the human kinome. The potency of TERN-701 against the proliferation of wild type and mutant CML cell lines, as well as a panel of 102 cancer cell lines, was assessed using CellTiter-Glo®. Synergy between TERN-701 and active site TKIs was assessed in vitro with interactions quantified using Bliss, combination, and curve-shift analyses. Prospective mutagenesis experiments were conducted by treating BaF/3 cells engineered to overexpress the BCR::ABL1 kinase with various concentrations of both TERN-701 and the TKI imatinib, followed by observation of clonal outgrowth of treated cells over time. We have previously shown that TERN-701 is a potent inhibitor of native CML cell lines such as K562. Here, we show that TERN-701 inhibited cell proliferation in various clinically relevant mutant CML cell lines (including T315I) with IC 50s ranging from 5 to 26 nM and 70 to 1200 nM against active site and myristoyl site mutations, respectively. TERN-701 was highly selective for BCR::ABL1, with no appreciable activity (>50%) against >450 purified kinase targets. Against a panel of cancer cell lines, TERN-701 was more selective than the comparator allosteric BCR::ABL1 inhibitor asciminib while maintaining similar potency. In vitro combination studies previously revealed that TERN-701 works synergistically with multiple TKIs in the non-mutant K562 cell line. Here, expanded studies using KCL22-s and BCR::ABL1-T315I mutant cell lines identified strong synergistic interactions between TERN-701 and active site TKIs, including ponatinib and dasatinib. Furthermore, prospective mutagenesis studies showed that the combination of TERN-701 and imatinib resulted in greater inhibition of the outgrowth of resistant clones relative to either compound alone. TERN-701 is a potent and highly selective allosteric inhibitor of BCR::ABL1 that can act synergistically with active site TKIs in vitro, even against the T315I gatekeeper mutation that confers resistance to all approved active site TKIs except ponatinib, which has known safety liabilities. These data support the continued development of TERN-701 for the treatment of CML using both monotherapy and combination approaches.

Biochemical and Structural Characterization of OvoATh2: A Mononuclear Nonheme Iron Enzyme from Hydrogenimonas thermophila for Ovothiol Biosynthesis.

Ovothiol A and ergothioneine are thiol-histidine derivatives with sulfur substitutions at the δ-carbon or ε-carbon of the l-histidine imidazole ring, respectively. Both ovothiol A and ergothioneine have protective effects on many aging-related diseases, and the sulfur substitution plays a key role in determining their chemical and biological properties, while factors governing sulfur incorporation regioselectivities in ovothiol and ergothioneine biosynthesis in the corresponding enzymes (OvoA, Egt1, or EgtB) are not yet known. In this study, we have successfully obtained the first OvoA crystal structure, which provides critical information to explain their C-S bond formation regioselectivity. Furthermore, OvoATh2 exhibits several additional activities: (1) ergothioneine sulfoxide synthase activity akin to Egt1 in ergothioneine biosynthesis; (2) cysteine dioxygenase activity using l-cysteine and l-histidine analogues as substrates; (3) cysteine dioxygenase activity upon mutation of an active site tyrosine residue (Y406). The structural insights and diverse chemistries demonstrated by OvoATh2 pave the way for future comprehensive structure-function correlation studies.

Increased binding of anti-dsDNA antibodies to short oligonucleotides modified with topoisomerase I reveals a potential new enzyme function independent from DNA relaxation

ObjectiveTopoisomerase I (topo I) is a highly conserved enzyme which is known to reduce torsional stress at double-stranded (ds) DNA. Torsional stress induced by supercoiling of dsDNA requires either very long dsDNA existing in genomic DNA or circulation as presented in plasmid DNA. To enable DNA relaxation, topo I induce a transient single-strand break followed by stress-relieving rotation of the released DNA strand. Our group found by serendipity that the topo I inhibitor irinotecan is able to suppress murine systemic lupus erythematosus (SLE), an autoimmune disease which is characterized by the existence of pathogenic anti-dsDNA antibodies (abs). As a possible mechanism we demonstrated in the absence of immunosuppression an increased binding of anti-dsDNA abs to long genomic or circulated plasmid dsDNA modified with topo I.ResultsHere we show that this effect requires active site tyrosine of topo I which is known to facilitate DNA relaxation activity. Moreover, topo I enhanced anti-dsDNA abs binding to short linear oligonucleotides down to a size of 42 bp. Since oligonucleotides of such length are devoid of torsional stress and relaxation respectively, our results suggest a new and unknown function for the enzyme topo I.

Oral Abstract: CML-593 The Novel BCR::ABL1 Allosteric Inhibitor HS-10382/TERN-701 is Potent Against Mutations Resistant to Active Site Tyrosine Kinase Inhibitors (TKIs) and Acts Synergistically With TKIs in BCR::ABL1+ Cancer Cell Lines

Oral Abstract: CML-593 The Novel BCR::ABL1 Allosteric Inhibitor HS-10382/TERN-701 is Potent Against Mutations Resistant to Active Site Tyrosine Kinase Inhibitors (TKIs) and Acts Synergistically With TKIs in BCR::ABL1+ Cancer Cell Lines

POSTER: CML-593 The Novel BCR::ABL1 Allosteric Inhibitor HS-10382/TERN-701 is Potent Against Mutations Resistant to Active Site Tyrosine Kinase Inhibitors (TKIs) and Acts Synergistically With TKIs in BCR::ABL1+ Cancer Cell Lines

POSTER: CML-593 The Novel BCR::ABL1 Allosteric Inhibitor HS-10382/TERN-701 is Potent Against Mutations Resistant to Active Site Tyrosine Kinase Inhibitors (TKIs) and Acts Synergistically With TKIs in BCR::ABL1+ Cancer Cell Lines

CML-593 The Novel BCR::ABL1 Allosteric Inhibitor HS-10382/TERN-701 is Potent Against Mutations Resistant to Active Site Tyrosine Kinase Inhibitors (TKIs) and Acts Synergistically With TKIs in BCR::ABL1+ Cancer Cell Lines

CML-593 The Novel BCR::ABL1 Allosteric Inhibitor HS-10382/TERN-701 is Potent Against Mutations Resistant to Active Site Tyrosine Kinase Inhibitors (TKIs) and Acts Synergistically With TKIs in BCR::ABL1+ Cancer Cell Lines

Extensive Bioinformatics Analyses Reveal a Phylogenetically Conserved Winged Helix (WH) Domain (Zτ) of Topoisomerase IIα, Elucidating Its Very High Affinity for Left-Handed Z-DNA and Suggesting Novel Putative Functions

The dynamic processes operating on genomic DNA, such as gene expression and cellular division, lead inexorably to topological challenges in the form of entanglements, catenanes, knots, “bubbles”, R-loops, and other outcomes of supercoiling and helical disruption. The resolution of toxic topological stress is the function attributed to DNA topoisomerases. A prominent example is the negative supercoiling (nsc) trailing processive enzymes such as DNA and RNA polymerases. The multiple equilibrium states that nscDNA can adopt by redistribution of helical twist and writhe include the left-handed double-helical conformation known as Z-DNA. Thirty years ago, one of our labs isolated a protein from Drosophila cells and embryos with a 100-fold greater affinity for Z-DNA than for B-DNA, and identified it as topoisomerase II (gene Top2, orthologous to the human UniProt proteins TOP2A and TOP2B). GTP increased the affinity and selectivity for Z-DNA even further and also led to inhibition of the isomerase enzymatic activity. An allosteric mechanism was proposed, in which topoII acts as a Z-DNA-binding protein (ZBP) to stabilize given states of topological (sub)domains and associated multiprotein complexes. We have now explored this possibility by comprehensive bioinformatic analyses of the available protein sequences of topoII representing organisms covering the whole tree of life. Multiple alignment of these sequences revealed an extremely high level of evolutionary conservation, including a winged-helix protein segment, here denoted as Zτ, constituting the putative structural homolog of Zα, the canonical Z-DNA/Z-RNA binding domain previously identified in the interferon-inducible RNA Adenosine-to-Inosine-editing deaminase, ADAR1p150. In contrast to Zα, which is separate from the protein segment responsible for catalysis, Zτ encompasses the active site tyrosine of topoII; a GTP-binding site and a GxxG sequence motif are in close proximity. Quantitative Zτ-Zα similarity comparisons and molecular docking with interaction scoring further supported the “B-Z-topoII hypothesis” and has led to an expanded mechanism for topoII function incorporating the recognition of Z-DNA segments (“Z-flipons”) as an inherent and essential element. We further propose that the two Zτ domains of the topoII homodimer exhibit a single-turnover “conformase” activity on given G(ate) B-DNA segments (“Z-flipins”), inducing their transition to the left-handed Z-conformation. Inasmuch as the topoII-Z-DNA complexes are isomerase inactive, we infer that they fulfill important structural roles in key processes such as mitosis. Topoisomerases are preeminent targets of anti-cancer drug discovery, and we anticipate that detailed elucidation of their structural–functional interactions with Z-DNA and GTP will facilitate the design of novel, more potent and selective anti-cancer chemotherapeutic agents.

Investigating molecular interaction between TDP1 and PARP1.

DNA topoisomerase is responsible for regulation of DNA topology. Topoisomerase I (topoI) cleaves and rejoins the DNA single strand by forming a transient covalent complex with DNA via a phosphodiester bond. Tyrosyl-phosphodiesterase 1 (TDP1) catalyzes the hydrolysis of the phosphodiester bond between an active site tyrosine in HtopoI and DNA 3’-phosphate making TDP1 functionally connected with HtopoI activity. N-terminal domain (NTD) of TDP1 interacts with C-terminal domain (CTD) of Poly [ADP-ribose] polymerase 1 (PARP1) and gets PARylated that enhances recruitment of TDP1 to DNA damage sites. Herein, we investigated molecular interaction of complex formation between TDP1 and PARP1. We modelled a full-length PARP1 and obtained the TDP1-PARP1 complex using molecular docking. Molecular dynamics simulations were utilized to study structural integrity of the predicted complex and to investigate the residues involved in the formation of the TDP1-PARP1 complex. Surface plasmon resonance (SPR)-based experimental approach resulted in a KD value of ∼5 nM for the binding between these two functionally important proteins.

A suicide diiron oxygenase in p‐aminobenzoate biosynthesis in Chlamydia trachomatis

Folate is an essential one‐carbon carrier cofactor required for essential biological reactions such as amino acid and DNA biosynthesis. Folates are comprised of three chemical moieties: a pteridine ring, p‐aminobenzoate (pABA), and glutamate residues. Chlamydia trachomatis has been shown to synthesize folate de novo; however, it is missing several genes involved in the canonical folate biosynthesis pathway. Previous work has demonstrated that a single gene, ct610, functionally replaces the enzymes responsible for pABA production in the canonical biosynthesis pathway. However, CT610 does not use chorismate as the precursor to pABA as is used in the canonical pathway. Further experiments showed that isotopically labeled tyrosine was incorporated into the pABA molecule when synthesized by CT610 in vivo. However, in vitro experiments demonstrated that CT610 produces pABA in the presence of oxygen and a reducing agent without any added substrates, including tyrosine. This led to the hypothesis that CT610 is a self‐sacrificing “suicide” enzyme that donates an active site tyrosine residue in synthesizing pABA. CT610 has low sequence similarity to non‐heme diiron monooxygenases such as methane monooxygenase and class Ia ribonucleotide reductase, and the previously solved crystal structure of CT610 revealed a diiron active site. Here we describe our recent progress towards understanding this unique suicide mechanism for pABA biosynthesis. To determine the tyrosine residue sacrificed in the reaction, we generated five active site tyrosine to phenylalanine mutants and found that two of the variants completely abolished pABA production, suggesting that one of these residues is the precursor to pABA. Further, a conserved lysine residue is also essential for the reaction and may be a sacrificial amino group donor. We have confirmed the oxygenase activity of wild‐type CT610, which requires only a reducing agent and is not stimulated by the presence of tyrosine or other possible substrates. Further, we developed an Fe(II) reconstitution procedure, where the reconstituted enzyme exhibits a drastic increase in oxygenase activity. However, surprisingly, the reconstituted enzyme does not exhibit increased pABA synthase activity, although EDTA treatment of the as‐purified enzyme almost completely inhibits pABA production. This suggests that a metal other than iron is required for the reaction and thus current work is focused on testing other potential metal cofactors that may play a role in the novel suicide reaction.

Mechanisms to Repair Stalled Topoisomerase II-DNA Covalent Complexes.

DNA topoisomerases regulate the topological state of DNA, relaxing DNA supercoils and resolving catenanes and knots that result from biologic processes, such as transcription and replication. DNA topoisomerase II (TOP2) enzymes achieve this by binding DNA and introducing an enzyme-bridged DNA double-strand break (DSB) where each protomer of the dimeric enzyme is covalently attached to the 5' end of the cleaved DNA via an active site tyrosine phosphodiester linkage. The enzyme then passes a second DNA duplex through the DNA break, before religation and release of the enzyme. However, this activity is potentially hazardous to the cell, as failure to complete religation leads to persistent TOP2 protein-DNA covalent complexes, which are cytotoxic. Indeed, this property of topoisomerase has been exploited in cancer therapy in the form of topoisomerase poisons which block the religation stage of the reaction cycle, leading to an accumulation of topoisomerase-DNA adducts. A number of parallel cellular processes have been identified that lead to removal of these covalent TOP2-DNA complexes, facilitating repair of the resulting protein-free DSB by standard DNA repair pathways. These pathways presumably arose to repair spontaneous stalled or poisoned TOP2-DNA complexes, but understanding their mechanisms also has implications for cancer therapy, particularly resistance to anti-cancer TOP2 poisons and the genotoxic side effects of these drugs. Here, we review recent progress in the understanding of the processing of TOP2 DNA covalent complexes, the basic components and mechanisms, as well as the additional layer of complexity posed by the post-translational modifications that modulate these pathways. SIGNIFICANCE STATEMENT: Multiple pathways have been reported for removal and repair of TOP2-DNA covalent complexes to ensure the timely and efficient repair of TOP2-DNA covalent adducts to protect the genome. Post-translational modifications, such as ubiquitination and SUMOylation, are involved in the regulation of TOP2-DNA complex repair. Small molecule inhibitors of these post-translational modifications may help to improve outcomes of TOP2 poison chemotherapy, for example by increasing TOP2 poison cytotoxicity and reducing genotoxicity, but this remains to be determined.

Ionic Atmosphere Effect on the Absorption Spectrum of a Flavoprotein: A Reminder to Consider Solution Ions.

This study utilizes the FMN-dependent NADH:quinone oxidoreductase from Pseudomonas aeruginosa PAO1 to investigate the effect of introducing an active site negative charge on the flavin absorption spectrum both in the absence and presence of a long-range electrostatic potential coming from solution ions. There were no observed changes in the flavin UV-visible spectrum when an active site tyrosine (Y277) becomes deprotonated in vitro. These results could only be reproduced computationally using average solvent electrostatic configuration (ASEC) QM/MM simulations that include both positive and negative solution ions. The same calculations performed with minimal ions to neutralize the total protein charge predicted that deprotonating Y277 would significantly alter the flavin absorption spectrum. Analyzing the distribution of solution ions indicated that the ions reorganize around the protein surface upon Y277 deprotonation to cancel the effect of the tyrosinate on the flavin absorption spectrum. Additional biochemical experiments were performed to test this hypothesis.

A suicide diiron oxygenase in p ‐aminobenzoate biosynthesis in Chlamydia trachomatis

Folate is an essential cofactor required for several processes including DNA and amino acid biosynthesis. Folate molecules are made up of three parts: a pteridine ring, p-aminobenzoate (pABA), and a variable number of glutamate residues. Chlamydia trachomatis synthesizes folate de novo; however, several genes encoding enzymes required for the canonical folate biosynthesis pathway are missing, including PabA/B and PabC, which are normally required for pABA biosynthesis from chorismate. Previous studies have found that a single gene in C. trachomatis, CT610, functionally replaces the canonical pABA biosynthesis genes. Interestingly, CT610 does not use chorismate as a substrate. Instead, the CT610-route for pABA biosynthesis incorporates isotopically-labeled tyrosine into the synthesized pABA molecule. However, in vitro experiments revealed that CT610 produces pABA without any added substrates (including tyrosine) in the presence of a reducing agent and molecular oxygen. CT610 shares low sequence similarity to non-heme diiron oxygenases and the previously solved crystal structure revealed a diiron active site. Taken together, CT610 is proposed to be a self-sacrificing “suicide” enzyme that uses one of its active site tyrosine residues as a precursor to pABA in a reaction that requires O2 and a reduced diiron cofactor. Here, we discuss our recent progress towards uncovering the mechanism of CT610-catalyed pABA synthesis.

The role of the active site tyrosine in the mechanism of lytic polysaccharide monooxygenase.

Catalytic breakdown of polysaccharides can be achieved more efficiently by means of the enzymes lytic polysaccharide monooxygenases (LPMOs). However, the LPMO mechanism has remained controversial, preventing full exploitation of their potential. One of the controversies has centered around an active site tyrosine, present in most LPMO classes. Recent investigations have for the first time obtained direct (spectroscopic) evidence for the possibility of chemical modification of this tyrosine. However, the spectroscopic features obtained in the different investigations are remarkably different, with absorption maximum at 420 and 490 nm, respectively. In this paper we use density functional theory (DFT) in a QM/MM formulation to reconcile these (apparently) conflicting results. By modeling the spectroscopy as well as the underlying reaction mechanism we can show how formation of two isomers (both involving deprotonation of tyrosine) explains the difference in the observed spectroscopic features. Both isomers have a [TyrO–Cu–OH]+ moiety with the OH in either the cis- or trans-position to a deprotonated tyrosine. Although the cis-[TyrO–Cu–OH]+ moiety is well positioned for oxidation of the substrate, preliminary calculations with the substrate reveal that the reactivity is at best moderate, making a protective role of tyrosine more likely.

Articles of Significant Interest in This Issue

Chlamydia trachomatis is missing the three genes normally required for p-aminobenzoate (pABA) biosynthesis. Previous genetic studies identified a single gene, the CT610 gene, responsible for pABA biosynthesis in C. trachomatis, but the molecular details of this unusual pathway remained unknown. Macias-Orihuela et al. (e00319-20) demonstrate that tyrosine is the precursor to the aromatic portion of pABA in the CT610-dependent route. Most interestingly, they further provide evidence that CT610 sacrifices its own active-site tyrosine residue(s) for pABA biosynthesis in a reaction that requires O2 and a reduced diiron cofactor. This novel “suicide” pABA synthase could be exploited as an innovative antibiotic target.

A Type IA DNA/RNA Topoisomerase with RNA Hydrolysis Activity Participates in Ribosomal RNA Processing

Topoisomerases maintain topological homeostasis of bacterial chromosomes by catalysing changes in DNA linking number. The resolution of RNA entanglements occurring in the cell would also require catalytic action of topoisomerases. We describe RNA topoisomerase and hydrolysis activities in DNA topoisomerase I (topo I) from mycobacteria. The interaction of topo I with mRNA, tRNA and rRNA suggested its role in some aspect of RNA metabolism; the enzyme participates in rRNA maturation via its RNA hydrolysis activity. Accumulation of rRNA precursors in a topo I knockdown strain and the rescue of rRNA processing deficiency in RNaseE knockdown cells by topo I expression indicated the enzyme’s back-up support to RNases involved in rRNA processing. We demonstrate that the active-site tyrosine of the enzyme mediates catalytic reactions with both DNA/RNA substrates, and RNA topoisomerase activity can follow two reaction paths in contrast to its DNA topoisomerase activity. Mutation in the canonical proton relay pathway impacts DNA topoisomerase activity whilst retaining activity on RNA substrates. The mycobacterial topo I thus exemplifies the resourcefulness and parsimony of biological catalysis in harnessing the limited chemical repertoire at its disposal to find common solutions to mechanistically related challenges of phosphodiester breakage/exchange reactions in DNA and RNA that are essential for cell survival.

Investigation of the binding interaction of α-amylase with Chrysophyllum albidum seed extract and its silver nanoparticles: A multi-spectroscopic approach

• α-Amylase binding interactions with CSME and CSAgNP were studied. • UV-vis, FT-IR and fluorescence showed evidence of binding interactions. • Intrinsic fluorescence of α-amylase was quenched by static mechanism. • Microenvironments of α-amylase active site tyrosine residues were altered. • CSME and CSAgNP showed strong inhibition against α-amylase. The interactions between α-amylase, one of the key enzymes linked with postprandial glucose regulation, and Chrysophyllum albidum seed methanolic extract(CSME) and its green-synthesized silver nanoparticles(CSAgNP) were investigated using multiple spectroscopy including Fourier Transform-Infrared(FT-IR), ultraviolet(UV)-visible absorption, fluorescence spectroscopy, and biochemical analysis. FT-IR spectroscopy revealed presence of some functional groups in the samples. CSME and CSAgNP inhibited α-amylase. The intrinsic fluorescence intensity of α-amylase was quenched by CSME and CSAgNP via static mechanisms, indicating formation of complex between the enzyme and inhibitors. α-Amylase-CSAgNP complex had higher binding constants. The binding processes were exothermic, entropy driven, spontaneous, and involved hydrogen bonds and van der Waals force. Synchronous fluorescence quenching indicated alteration in microenvironment of α-amylase catalytic site tyrosine residues.FT-IR spectroscopy revealed shifts in amide I peak position of α-amylase due to interaction with CSME/CSAgNP. Absorption spectroscopy also affirmed changes in enzyme conformation. This study may provide theoretical basis for designing novel α-amylase inhibitors. The binding interactions of α-amylase with CSME and CSAgNP were investigated/ affirmed using multiple spectroscopic and biochemical methods following established protocols.